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Glidden Thesis FINAL (3-28-18).pdf (12.47 MB)

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Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs

Glidden, Michael D., II
http://orcid.org/0000-0003-3069-8908
http://rave.ohiolink.edu/etdc/view?acc_num=case1522270994798884

Abstract Details

2018, Doctor of Philosophy, Case Western Reserve University, Physiology and Biophysics.
Single-chain insulin (SCI) analogs have long provided a valuable model for structure-function studies of insulin. Recently, a biologically active SCI containing a six-residue connecting (C) domain (inserted between the B- and A chains of two-chain insulin) demonstrated marked resistance to thermal degradation. Such a design has potential to circumvent the cold-storage requirements of current insulin therapeutic formulations which hinder their distribution, storage, and use in developing regions. An understanding of how this SCI scaffold can be engineered to confer desired biophysical and pharmacological properties would enable rational design, development, and clinical implementation of SCIs. This Thesis leverages principles of protein folding, structure, dynamics, stability and function to modulate SCI properties. Following an introduction of insulin analog design with focus on how SCIs could address a global need for ultra-stable formulations (Chapter 1), a synthetic single-chain [des-B29,des-B30]-insulin platform was utilized to model an oxidative intermediate in the folding of wild-type insulin. This system enabled comparative studies of an unstable TyrB16->Pro variant, which exhibited a severe folding defect (Chapter 2). Chapters 3-4 contain studies of two novel biologically active SCIs: SCI-a and SCI-b. These analogs exhibited remarkable resistance to thermal inactivation; SCI-a had native-like duration of hormone action in diabetic rats. Collaborative X-ray crystallographic studies of SCI-a revealed a novel zinc-free T6 hexamer (Chapter 3) whereas NMR and molecular dynamics studies of SCI-b provided insight into its mechanisms of enhanced stability and receptor binding (Chapter 4). Studies of variant SCIs containing amino-acid substitutions at position A14 (Chapter 5) suggested that the reverse hydrophobic effect, a general biophysical principle uncommonly observed, was operative. This principle could be leveraged to enhance SCI resistance to chemical degradation. An unexpected prolongation of hormone action observed upon intravenous injection of SCI-b in diabetic rats (Chapters 3 and 4) motivated mutagenesis of A- and C domain sites (Chapter 6). Our results identified a set of novel fast-acting SCIs. A Thesis summary (Chapter 7) is followed by a discussion of how the findings presented herein may motivate future biophysical studies. Implications for the development of SCI therapeutic formulations and their potential impact on global diabetes care are discussed.
Michael Weiss, MD, PhD (Advisor)
Matthias Buck, DPhil (Committee Chair)
Faramarz Ismail-Beigi, MD, PhD (Committee Member)
George Dubyak, PhD (Committee Member)
Michael Zagorski, PhD (Committee Member)
390 p.
Biochemistry; Biophysics
single-chain insulin analog; hormone; diabetes mellitus; protein engineering; protein stability; protein structure and folding; protein dynamics; nuclear magnetic resonance; single-chain insulin pharmacology

Recommended Citations

Citations

  • Glidden, II, M. D. (2018). Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1522270994798884

    APA Style (7th edition)

  • Glidden, II, Michael. Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs. 2018. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1522270994798884.

    MLA Style (8th edition)

  • Glidden, II, Michael. "Single-chain insulin analogs as ultra-stable therapeutics and as models of protein (mis)folding: stability, structure, dynamics, and function of novel analogs." Doctoral dissertation, Case Western Reserve University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1522270994798884

    Chicago Manual of Style (17th edition)

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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.